PROJECT SUMMARY/ABSTRACT Hedgehog (Hh) signaling is essential for tissue patterning and cell proliferation in development, regeneration, and disease. As classical morphogens, Hh ligands direct cell fate decisions in a manner dependent on signaling strength. Precise regulation of signaling strength is critical for proper tissue development and maintenance, highlighted by the fact that even modest changes in the signaling amplitude can result in human birth defects. While a large body of work has shown that signaling strength is influenced by the concentration and exposure time of Hh ligands, an equally important layer of regulation remains unrecognized and unstudied: How is the sensitivity of target cells to morphogens regulated? During the first phase of my postdoctoral training, I used genome-wide CRISPR screens to discover three genes that function to attenuate Hh signaling in target cells: Mosmo, Megf8, and Mgrn1 (the ?MMM module?). Disruption of the MMM module in both fibroblasts and neural progenitor cells (NPCs) resulted in a ~10-fold increase in sensitivity to Hh ligands and altered neural cell-fate decisions, driven by a marked increase in levels of the transmembrane transducer Smoothened (SMO) at primary cilia. These studies lead to the hypothesis that the MMM module regulates signaling strength in target cells by regulating the sub-cellular localization of Hh pathway components. To test this hypothesis, I will (1) determine the mechanism by which the MMM module regulates SMO trafficking, (2) illuminate the role of ubiquitination in MMM-regulated SMO trafficking and Hh sensitivity, and (3) identify the function of the MMM module during embryonic development. These studies will unravel the molecular basis and physiological function of a novel mechanism that allows target cells to modify their responses to extracellular cues and consequently suggest new strategies to modulate Hh signaling strength in disease states. In graduate school, I trained as a mouse geneticist and embryologist. During the first phase of my postdoc, I learned how to use CRISPR technology to conduct genome-wide loss-of-function screens and to test the function of specific genes in sophisticated in vitro differentiation assays. Support from the K99 program, the resources available at Stanford University, and the expertise of my advisory panel will allow me to develop critical new skills in the areas of advanced microscopy, protein biochemistry, and mass spectrometry to understand the biochemical and biological function of developmental regulators like the MMM module. I will accomplish this with training from my mentor Dr. Rajat Rohatgi (biochemistry and cancer biology), my co- mentor Dr. Tim Stearns (cilia biology), and a strong advisory panel composed of members with expertise in protein trafficking, computational biology, developmental biology, and mass spectrometry. The training and mentorship I receive during my K99/R00 award will provide a critical stepping stone for me to achieve my academic goal of establishing a vibrant independent research program that can answer important questions in developmental signaling using approaches ranging from mouse genetics to mechanistic biochemistry.